G Model
JINJ-5672; No. of Pages 5 Injury, Int. J. Care Injured xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Injury journal homepage: www.elsevier.com/locate/injury
Sphingosine kinase 1 improves cutaneous wound healing in diabetic rats Hongyang Yu a,c, Lizhen Yuan a, Mingbo Xu b, Zhenqing Zhang c, Haifeng Duan a,* a
Beijing Institute of Radiation Medicine (BIRM), No. 27, Taiping Road, Beijing 100850, Haidian District, China Beijing Shuanglu Pharmaceutical Company, No. 69, Fushi Road, Beijing 100049, Haidian District, China c Beijing Institute of Pharmacology and Toxicology, No. 27, Taiping Road, Beijing 100850, Haidian District, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Accepted 3 March 2014
Background: Diabetes is one of the most prevalent human metabolic diseases. Wound healing in diabetes is frequently impaired and treatment remains challenging. Sphingolipid metabolites play important roles in the regulation of glucose metabolism. SPK1 is the key enzyme in the sphingolipid metabolic pathway. S1P/ SPK plays a pivotal role in the signalling pathways of diverse cellular processes including proliferation, differentiation, migration, apoptosis in diverse cell types. Methods: To investigate the role of sphingosine kinase 1 (SPK1) in skin injury, plasmids containing the SPK1 gene (pcDNA3-FLAG-SPK1) were applied to cutaneous wounds on a streptozotocin-induced diabetic rat model over a 21-day period. The wound area and rate of wound healing were determined. The histopathological features of the healed wounds were also observed, and SPK1 expression in the skin was detected by immunohistochemistry. Results: There was a significant decrease in wound area in diabetic rats treated with 125 and 60 mg/ wound pcDNA3-FLAG-SPK1 (P < 0.001–0.01). The mean sizes of the wounds were 0.67 0.15 cm2, 0.83 0.18 cm2, and 1.09 0.23 cm2 in both treated and diabetic control group at the 7th day posttreatment respectively. In addition, wound healing in diabetic rats of test group was accelerated. At the 7th day, the mean rates of healing were 73.2 5.7% and 66 7.3% in test group of 125 and 60 mg/wound respectively, and 55.4 9.9% in diabetic control group (P < 0.001–0.01). Histology revealed that tissue sections from the treated diabetic rats contained more granulation tissue and capillaries than that of the control rats. There was high SPK1 expression in the skin of the treated diabetic rats. Conclusions: SPK1 gene therapy may represent a novel approach to cutaneous wound healing. ß 2014 Elsevier Ltd. All rights reserved.
Keywords: Sphingosine kinase 1 Diabetic wound healing Diabetic rats
Introduction Diabetes is one of the most prevalent human metabolic diseases, reaching pandemic proportions [1]. The morbidity of diabetes has increased dramatically over the past few years, and afflicts more than 100 million people worldwide. Delayed wound healing is considered one of the most common, disabling, and costly complications of diabetes. People with diabetes have poor circulation, poor resistance to infection, and poor local nutrition, thus their wounds are highly susceptible to infection [2]. This impairment in diabetic wound healing represents a significant clinical problem, leading to chronic non-healing ulcers ultimately resulting in infection, gangrene, or even amputation [3]. Thus, accelerating wound healing must be the primary goal of wound care.
* Corresponding author. Tel.: +86 10 66930230; fax: +86 10 68158312. E-mail address: [email protected] (H. Duan).
Sphingosine kinase (SPK), a highly conserved lipid kinase present in organisms as diverse as humans, mice, flies, yeast, and plants, catalyses the phosphorylation of sphingosine to generate sphingosine-1-phosphate (S1P), a potent lipid mediator that plays important roles in a wide variety of mammalian cellular processes. SPK1 and SPK2 are the only two cloned isoforms of mammalian SPK. SPK1 is located predominantly in the cytosol; small amounts are associated with cellular membranes [4]. Sphingolipid metabolites play important roles in the regulation of glucose metabolism. SPK1 is the key enzyme in the sphingolipid metabolic pathway, forming an essential checkpoint to regulate the relative levels of bioactive sphingolipid metabolites, ceramide, sphingosine, and S1P. Previously [5], we demonstrated that adenovirusmediated SPK1 gene transfer (Ad-SPK1) improved blood glucose and lipid profiles, reduced body weight and adiposity, reversed hepatic steatosis, increased energy expenditure, and improved insulin sensitivity in diabetic mice. In this study, we investigated the effect of SPK1 on wound healing in diabetes mellitus.
http://dx.doi.org/10.1016/j.injury.2014.03.003 0020–1383/ß 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yu H, et al. Sphingosine kinase 1 improves cutaneous wound healing in diabetic rats. Injury (2014), http://dx.doi.org/10.1016/j.injury.2014.03.003
G Model
JINJ-5672; No. of Pages 5 2
H. Yu et al. / Injury, Int. J. Care Injured xxx (2014) xxx–xxx
SPK1 catalyses the phosphorylation of sphingosine to form S1P [6]. An increasing amount of evidence indicates that S1P/SPK plays a pivotal role in the signalling pathways of diverse cellular processes in various cell types, including proliferation, differentiation, migration, and apoptosis [7–10]. SPK1 also promotes cell growth in soft agar [11]. After demonstrating markedly improved blood glucose levels in type 2 diabetic KK/Ay mice by Ad-SPK1, we designed the present study to assess the effect of SPK1 on wound healing in diabetic rats.
after wounding. The wound size was then calculated by weighing the film. Animals were sacrificed 7, 14, and 21 days after wounding and skin specimens were obtained and fixed in 10% paraformaldehyde for histological study. Rate of wound healing
Materials and methods
The percentage of wound closure was calculated as follows, using the initial and final wound area drawn on glass slides during the experiments: % of wound healing = (original area unhealed area)/original area 100%.
Animals and plasmid
Histopathological assay
Healthy adult male Wistar rats (approximately 10 weeks of age; average body weight of 250–300 g) were used in this study. All rats were housed at a constant temperature and humidity in a room with an artificial 12-h light/dark cycle and had free access to food and water. Constructs of wild-type SPK1 (pcDNA3-FLAG-SPK1) were kindly provided by Dr. Stuart M. Pitson (Hanson Institute, Adelaide, Australia) [12]. All animal experiments were conducted following the institutional guidelines and approved by the Ethical Committee for Animal Care and Use, The Military Medical Science Academy of the PLA.
A skin specimen was obtained from each group on day 7, 14, 21 after wounding for histopathological examination. Specimens were immediately fixed in 10% (v/v) neutral buffered formalin, and the solution was replaced every 2 days until the tissues had hardened. Each specimen was embedded in a paraffin block and thin sections (3 mm) were prepared and stained with haematoxylin and eosin (H&E) for general morphological observation.
Reagent and antibody Streptozotocin (STZ) was purchased from Sigma (St. Louis, MO, USA). Rabbit anti-FLAG antibody was obtained from OriGene (Rockville, MD, USA). Induction of diabetes and wounding Forty male rats were used; five were randomly selected as normal controls. Diabetes was induced in the remaining rats by tail intravenous injection of the pancreatic b-cell toxin STZ (freshly dissolved in sterile saline, 2%) at a dose of 50 mg/kg body weight. Blood glucose levels were measured using an acute glucometer (Roche, USA) 1 week after the STZ injections. Animals that developed average blood glucose concentrations exceeding 18 mmol/L were selected and assigned randomly to four groups. Wounding was performed as described previously [13]. Briefly, rats were anaesthetised with intraperitoneal Nembutal (50 mg/ kg). The dorsal skin was shaved and cleaned with povidone-iodine solution. Two full-thickness skin wounds (approximately 1.8-cm diameter) were created on the back of each rat: the area was marked with a stamp before the outlined skin was cut. Therapeutic intervention and wound area Different concentrations of plasmid-SPK1 constructs (pcDNA3FLAG-SPK1; 15, 30, 60, 120 mg/wound) were applied to the wounds every 3 days. The construct was not applied to rats in the diabetic control group. Wound size was recorded with a transparent film after anaesthesia at 0, 3, 7, 10, 14, and 21 days
Immunohistochemistry Tissue sections that had been dewaxed and rehydrated routinely were incubated with 3% H2O2 for 30 min. The slides were washed with phosphate-buffered saline (PBS, pH 7.4) twice. The sections were blocked with 5% bovine serum albumin in Trisbuffered saline for 20 min. The spent solution was discarded and the sections were incubated with anti-FLAG antibody (1:2000) at 4 8C overnight. The slides were washed with PBS, incubated with rabbit secondary antibody (1:5000) for 1 h, and followed by incubation with streptavidin–horseradish peroxidase for 20 min. The antibody binding sites were visualised by incubation with diaminobenzidine–H2O2 solution. Statistical analysis All values are expressed as mean standard error of the mean (SEM). One-way analysis of variance followed by Student’s unpaired t-test was used to compare parametric data. P < 0.05 was recognised to indicate statistical significance. Results Effect of SPK1 on blood glucose levels of diabetic rats There was a significant elevation in blood glucose level (>18 mmol/L) after 1 week in the rats that had received STZ. Blood glucose levels were determined when the plasmid treatment was administered, as shown in Table 1; all diabetic rats had average blood glucose levels that exceeded 18 mmol/L, although there was no statistically significant difference among the six groups. Our data indicate that SPK1 did not directly affect the blood glucose levels of diabetic rats.
Table 1 Effect of SPK1 on blood glucose levels of diabetic rats. Group
Dose mg/wound
Times
Control Diabetic control SPK1 SPK1 SPK1 SPK1
Physiological saline Physiological saline 120 60 30 15
3 3 3 3 3 3
Blood glucose (mmol/L) 0d
7d
14 d
21 d
5.6 0.3 20.3 2.7 20.6 4.5 19.9 0.07 20.2 2.4 19.9 2.9
5.8 0.5 26.2 4.0 24.9 54.2 27.8 5.8 28.9 4.2 26.2 6.1
5.1 0.2 30.7 5.4 25.7 2.4 31.3 3.0 28.8 5.3 27.0 6.4
6.4 0.5 31.0 1.3 27.4 2.5 26.9 0.8 28.2 2.9 25.4 7.2
Please cite this article in press as: Yu H, et al. Sphingosine kinase 1 improves cutaneous wound healing in diabetic rats. Injury (2014), http://dx.doi.org/10.1016/j.injury.2014.03.003
G Model
JINJ-5672; No. of Pages 5 H. Yu et al. / Injury, Int. J. Care Injured xxx (2014) xxx–xxx
Fig. 1. Effect of SPK1 on wound areas of diabetic rats. Wound size was recorded with transparent film after anaesthesia at 0, 3, 7, 10, 14, and 21 days after wounding. The wound size was calculated by weighing the transparent films. Each bar represents the mean SEM wound area of each group. *P < 0.05, **P < 0.01, #P < 0.001 vs. diabetic control group.
Effect of SPK1 on wound areas of diabetic rats Wounds in all groups decreased in size gradually over time (Fig. 1). However, there was an obvious delay in the rate of healing in diabetic rats as compared with normal control rats. The wound areas of the diabetic rats were larger than that of the normal control rats after 3–21 days. Observation of the diabetic rats at 7 days revealed a statistically significant decrease in the wound area following treatment with 125 mg/wound (0.67 0.15 cm2, P < 0.001) and 60 mg/wound (0.83 0.18 cm2, P < 0.01) pcDNA3FLAG-SPK1 compared with diabetic control group (1.09 0.23 cm2). However, there was no significant change to the wound area at 3 days following treatment with 30 mg/wound pcDNA3-FLAG-SPK1 until day 7 of the treatment period, when a significant difference was observed (P < 0.05). No significant difference was detected when the dosage was reduced to 15 mg/wound. Effect of SPK1 on rate of wound healing in diabetic rats There was an obvious improvement in both wound area and rate of wound healing following treatment with different doses of pcDNA3-FLAG-SPK1 (Figs. 1 and 2). There was satisfactory healing following treatment with 125 and 60 mg/wound doses from 3 to 21 days. There was a statistically significant difference between the treatment and control groups (P < 0.05). After 21 days, the mean rate of healing was 98%. When a 30 mg/wound dose was administered, the initial effectiveness was delayed. When the dosage was reduced to 15 mg/wound, there was no effective healing. All groups had healed fully after 21 days. The results demonstrated that at a certain dosage range, SPK1 could accelerate wound healing in diabetic rats; the initial effective dose was 30 mg/ wound. A higher dose and earlier administration was followed by a higher rate of healing, which demonstrates an obvious dose–effect relationship. Effect of SPK1 on histopathological features of wounds of diabetic rats Following H&E staining (Fig. 3), the histopathological characteristics of the wounds on day 7, 14, and 21 after wounding were observed. After 7 days, control group specimens contained necrotic tissue and necrotic inflammatory cells, a small quantity of granulation tissue hyperplasia, and absence of re-epithelialisation (Fig. 3A). Conversely, tissue from the treatment groups (Fig. 3B) contained a large quantity of granulation tissue and abundant capillaries. After 14 days, the control group exhibited poor hyperplasia of granulation tissue accompanied by oedema and thinner re-epithelialisation at the edge of the wound with necrosis (Fig. 3C). Specimens from the treatment groups (Fig. 3D) contained
3
Fig. 2. Effect of SPK1 on rate of wound healing in diabetic rats. The percentage of wound closure was calculated by using the initial and final wound area as outlined on glass slides during the treatment period. Each bar represents the mean SEM wound healing rate of each group. *P < 0.05, **P < 0.01, #P < 0.001 vs. diabetic control group.
granulation tissue that had changed to fibrous connective tissue, and there were abundant capillaries. There was apparent reepithelialisation at the edge of the wound, which extended to cover the wound. After 21 days, control group specimens contained granulation tissue that had changed into fibrous connective tissue accompanied by oedema, and there was apparent re-epithelialisation that extended to cover the wound, but the tissue structure was incomplete (Fig. 3E). However, treatment groups specimens (Fig. 3F) showed that the granulation tissue had completely changed into fibrous connective tissue and the cutaneous wound had healed completely in most of the rats in the experiment. Detection of SPK1 expression in wound areas We detected the expression of SPK1 in the rat skins by immunohistochemistry (Fig. 4). Immunohistochemical staining with FLAG antibody demonstrated that there was high expression of SPK1 in the treatment groups, indicating that SPK1 was expressed on the surfaces of the wounds. Discussion Impaired wound healing, a common complication of diabetes mellitus, is characterised by diminished collagen production and impaired angiogenesis [14]. Wound healing is a complex multifactorial process involving inflammation, migration of different cell types, fibroplasia, collagen deposition, and wound contraction that results in the contraction and closure of the wound and restoration of a functional barrier [3]. Diabetes mellitus is one of the major contributors to chronic wound healing problems, as minor skin wounds can develop into chronic, nonhealing ulcers, ultimately resulting in infection, gangrene, or even amputation [3]. SPK is an enzyme that catalyses the phosphorylation of sphingosine to S1P. Zhang et al. demonstrated that exogenously applied sphingosine is rapidly taken up by cells and phosphorylated on its primary hydroxyl group into S1P [15]. Exogenous sphingosine can stimulate cell proliferation in several cell types [16,17]. This mitogenic effect of sphingosine may be mediated by its conversion to S1P, as S1P itself is an even more potent mitogen than sphingosine [15]. S1P is a sphingolipid metabolite present in high concentrations in the blood and is implicated in vascular development. It is secreted most prominently by platelets, suggesting that it plays an important role in tissue repair. Further, it has been shown to act via distinct receptor pathways to regulate keratinocyte and fibroblast chemotaxis, processes that are critical for normal wound healing [18,19]. As proof of concept,
Please cite this article in press as: Yu H, et al. Sphingosine kinase 1 improves cutaneous wound healing in diabetic rats. Injury (2014), http://dx.doi.org/10.1016/j.injury.2014.03.003
G Model
JINJ-5672; No. of Pages 5 4
H. Yu et al. / Injury, Int. J. Care Injured xxx (2014) xxx–xxx
Fig. 3. Histological examination of H&E-stained tissue sections. Photomicrographs depict tissue sections of the wounds on rats in the control and treatment groups on days 7, 14, and 21 after wounding. (A) Control group after 7 days; (B) treatment group after 7 days; (C) control group after 14 days; (D) treatment group after 14 days; (E) control group after 21 days; (F) treatment group after 21 days (HE stain, 200).
Fig. 4. Expression of SPK1 in cutaneous wounds of diabetic rats on day 7: (A) control group and (B) treatment group (200 magnification).
subcutaneous injections of S1P significantly improved diabetic wound healing and neovascularisation in rodent models [20]. We constructed a plasmid containing SPK1 cDNA and applied it to wound surfaces to examine its effect on wound healing, and found that SPK1 promoted wound healing. We hypothesised that SPK1 catalysed the phosphorylation of sphingosine to S1P in the skin. It is possible that this is a new approach to cutaneous wound repair. It is noteworthy that enhanced wound healing by
SPK1 cDNA application in diabetic rats is independent of blood glucose control. As shown in Table 1, there was no statistically significant difference in the blood glucose levels between the treated and control diabetic animals. Cutaneous wound healing is a complex pathological process in which a number of tissues, extracellular matrix, and cytokines are involved, and includes a series of biological processes. A key event in the process is hyperplasia of granulation tissue. The formation of
Please cite this article in press as: Yu H, et al. Sphingosine kinase 1 improves cutaneous wound healing in diabetic rats. Injury (2014), http://dx.doi.org/10.1016/j.injury.2014.03.003
G Model
JINJ-5672; No. of Pages 5 H. Yu et al. / Injury, Int. J. Care Injured xxx (2014) xxx–xxx
granulation tissue in a wound can repair tissue defects, enhance resistance of the wound to infection, and promote contraction so that the wound heals, creating the necessary conditions for epithelial climb. Granulation tissue is mainly composed of fibroblasts and new capillaries. Angiogenesis plays an essential role in the processes of organisation healing [21]. The proliferation of capillary endothelial cells and fibroblast cells can significantly promote the proliferation of granulation tissue and secrete many functional proteins while promoting wound contraction and expansion. The quantity of capillaries and fibroblasts determine the duration of wound healing. In the present study, histopathological examination revealed rapid re-epithelialisation and promotion of granulation tissue as well as blood vessel growth following the application of plasmids that carried the SPK1 gene to the skin around the wound. Located in the dermis layer, fibroblasts are one of the major repair cells. Wound repair depends on the biological behaviour of fibroblasts. The process of wound healing is a highly orchestrated event that entails the release of proinflammatory factors and growth factors. Once injury occurs, fibroblasts activate and proliferate in the early stages after wounding, becoming myofibroblasts which express smooth muscle actin (SMA). These newly formed cells participate in the secretion of many factors, wound contraction, and extracellular matrix production. With continuous wound repair, SMA expression is decreased and myofibroblasts gradually disappear, and the expression of various growth factors gradually decreases [22,23]. Therefore, myofibroblasts play a central role in wound healing. The involvement of SPK1 in wound healing effected by fibroblasts requires further investigation. Conclusions SPK1 had an obvious effect on wound healing in diabetes mellitus, accelerating wound healing in diabetic rats. There was an obvious improvement in both wound area and rate of wound healing following treatment with different doses of pcDNA3-FLAGSPK1. However, the treatment did not affect the blood glucose levels of the diabetic rats. Conflict of interest statement There are no conflicts of interest. Acknowledgements We thank Yonghong Lei for his help with data analysis. We thank Xiaoyu Zhu for her valuable help in the last phase of this study.
5
References [1] Leahy JL. Pathogenesis of type 2 diabetes mellitus. Arch Med Res 2005;36:197– 209. [2] Jahangiri Noudeh Y, Shabani M, Vatankhah N, Hashemian SJ, Akbari K. A combination of 670 nm and 810 nm diode lasers for wound healing acceleration in diabetic rats. Photomed Laser Surg 2010;28:621–7. [3] Lee YH, Chang JJ, Chien CT, Yang MC, Chien HF. Antioxidant sol–gel improves cutaneous wound healing in streptozotocin-induced diabetic rats. Exp Diabetes Res 2012;2012:504693. [4] Spiegel S, Milstien S. Sphingosine-1-phosphate: an enigmatic signalling lipid. Nat Rev Mol Cell Biol 2003;4:397–407. [5] Duan HF, Wu CT, Lu Y, Wang H, Liu HJ, Zhang QW, et al. Sphingosine kinase activation regulates hepatocyte growth factor induced migration of endothelial cells. Exp Cell Res 2004;298:593–601. [6] Nava VE, Lacana E, Poulton S, Liu H, Sugiura M, Kono K, et al. Functional characterization of human sphingosine kinase-1. FEBS Lett 2000;473:81–4. [7] Pyne S, Pyne NJ. Sphingosine 1-phosphate signalling in mammalian cells. Biochem J 2000;349:385–402. [8] Xia P, Gamble JR, Wang L, Pitson SM, Moretti PA, Wattenberg BW, et al. An oncogenic role of sphingosine kinase. Curr Biol 2000;10:1527–30. [9] Spiegel S, Milstien S. Sphingosine 1-phosphate, a key cell signaling molecule. J Biol Chem 2002;277:25851–54. [10] Olivera A, Kohama T, Edsall L, Nava V, Cuvillier O, Poulton S, et al. Sphingosine kinase expression increases intracellular sphingosine-1-phosphate and promotes cell growth and survival. J Cell Biol 1999;147:545–58. [11] Olivera A, Spiegel S. Sphingosine kinase: a mediator of vital cellular functions. Prostaglandins 2001;64:123–34. [12] Pitson SM, Moretti PA, Zebol JR, Xia P, Gamble JR, Vadas MA, et al. Expression of a catalytically inactive sphingosine kinase mutant blocks agonist-induced sphingosine kinase activation. A dominant-negative sphingosine kinase. J Biol Chem 2000;275:33945–50. [13] Mori R, Kondo T, Ohshima T, Ishida Y, Mukaida N. Accelerated wound healing in tumor necrosis factor receptor p55-deficient mice with reduced leukocyte infiltration. FASEB J 2002;16:963–74. [14] Hansen SL, Myers CA, Charboneau A, Young DM, Boudreau N. HoxD3 accelerates wound healing in diabetic mice. Am J Pathol 2003;163:2421–31. [15] Zhang H, Desai NN, Olivera A, Seki T, Brooker G, Spiegel S. Sphingosine-1phosphate, a novel lipid, involved in cellular proliferation. J Cell Biol 1991;114:155–67. [16] Zhang H, Buckley NE, Gibson K, Spiegel S. Sphingosine stimulates cellular proliferation via a protein kinase C-independent pathway. J Biol Chem 1990;265:76–81. [17] Merrill Jr AH, Schmelz EM, Dillehay DL, Spiegel S, Shayman JA, Schroeder JJ, et al. Sphingolipids – the enigmatic lipid class: biochemistry, physiology, and pathophysiology. Toxicol Appl Pharmacol 1997;142:208–25. [18] Estrada R, Zeng Q, Lu H, Sarojini H, Lee JF, Mathis SP, et al. Up-regulating sphingosine 1-phosphate receptor-2 signaling impairs chemotactic, woundhealing, and morphogenetic responses in senescent endothelial cells. J Biol Chem 2008;283:30363–75. [19] Vogler R, Sauer B, Kim DS, Schafer-Korting M, Kleuser B. Sphingosine-1phosphate and its potentially paradoxical effects on critical parameters of cutaneous wound healing. J Invest Dermatol 2003;120:693–700. [20] Kawanabe T, Kawakami T, Yatomi Y, Shimada S, Soma Y. Sphingosine 1phosphate accelerates wound healing in diabetic mice. J Dermatol Sci 2007;48:53–60. [21] Isner JM, Losordo DW. Therapeutic angiogenesis for heart failure. Nat Med 1999;5:491–2. [22] Powell DW, Mifflin RC, Valentich JD, Crowe SE, Saada JI, West AB, et al. Paracrine cells important in health and disease. Am J Physiol 1999;277:C1–9. [23] Badid C, Vincent M, Fouque D, Laville M, Desmouliere A. Myofibroblast: a prognostic marker and target cell in progressive renal disease. Ren Fail 2001;23:543–9.
Please cite this article in press as: Yu H, et al. Sphingosine kinase 1 improves cutaneous wound healing in diabetic rats. Injury (2014), http://dx.doi.org/10.1016/j.injury.2014.03.003
JINJ-5672; No. of Pages 5 Injury, Int. J. Care Injured xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Injury journal homepage: www.elsevier.com/locate/injury
Sphingosine kinase 1 improves cutaneous wound healing in diabetic rats Hongyang Yu a,c, Lizhen Yuan a, Mingbo Xu b, Zhenqing Zhang c, Haifeng Duan a,* a
Beijing Institute of Radiation Medicine (BIRM), No. 27, Taiping Road, Beijing 100850, Haidian District, China Beijing Shuanglu Pharmaceutical Company, No. 69, Fushi Road, Beijing 100049, Haidian District, China c Beijing Institute of Pharmacology and Toxicology, No. 27, Taiping Road, Beijing 100850, Haidian District, China b
A R T I C L E I N F O
A B S T R A C T
Article history: Accepted 3 March 2014
Background: Diabetes is one of the most prevalent human metabolic diseases. Wound healing in diabetes is frequently impaired and treatment remains challenging. Sphingolipid metabolites play important roles in the regulation of glucose metabolism. SPK1 is the key enzyme in the sphingolipid metabolic pathway. S1P/ SPK plays a pivotal role in the signalling pathways of diverse cellular processes including proliferation, differentiation, migration, apoptosis in diverse cell types. Methods: To investigate the role of sphingosine kinase 1 (SPK1) in skin injury, plasmids containing the SPK1 gene (pcDNA3-FLAG-SPK1) were applied to cutaneous wounds on a streptozotocin-induced diabetic rat model over a 21-day period. The wound area and rate of wound healing were determined. The histopathological features of the healed wounds were also observed, and SPK1 expression in the skin was detected by immunohistochemistry. Results: There was a significant decrease in wound area in diabetic rats treated with 125 and 60 mg/ wound pcDNA3-FLAG-SPK1 (P < 0.001–0.01). The mean sizes of the wounds were 0.67 0.15 cm2, 0.83 0.18 cm2, and 1.09 0.23 cm2 in both treated and diabetic control group at the 7th day posttreatment respectively. In addition, wound healing in diabetic rats of test group was accelerated. At the 7th day, the mean rates of healing were 73.2 5.7% and 66 7.3% in test group of 125 and 60 mg/wound respectively, and 55.4 9.9% in diabetic control group (P < 0.001–0.01). Histology revealed that tissue sections from the treated diabetic rats contained more granulation tissue and capillaries than that of the control rats. There was high SPK1 expression in the skin of the treated diabetic rats. Conclusions: SPK1 gene therapy may represent a novel approach to cutaneous wound healing. ß 2014 Elsevier Ltd. All rights reserved.
Keywords: Sphingosine kinase 1 Diabetic wound healing Diabetic rats
Introduction Diabetes is one of the most prevalent human metabolic diseases, reaching pandemic proportions [1]. The morbidity of diabetes has increased dramatically over the past few years, and afflicts more than 100 million people worldwide. Delayed wound healing is considered one of the most common, disabling, and costly complications of diabetes. People with diabetes have poor circulation, poor resistance to infection, and poor local nutrition, thus their wounds are highly susceptible to infection [2]. This impairment in diabetic wound healing represents a significant clinical problem, leading to chronic non-healing ulcers ultimately resulting in infection, gangrene, or even amputation [3]. Thus, accelerating wound healing must be the primary goal of wound care.
* Corresponding author. Tel.: +86 10 66930230; fax: +86 10 68158312. E-mail address: [email protected] (H. Duan).
Sphingosine kinase (SPK), a highly conserved lipid kinase present in organisms as diverse as humans, mice, flies, yeast, and plants, catalyses the phosphorylation of sphingosine to generate sphingosine-1-phosphate (S1P), a potent lipid mediator that plays important roles in a wide variety of mammalian cellular processes. SPK1 and SPK2 are the only two cloned isoforms of mammalian SPK. SPK1 is located predominantly in the cytosol; small amounts are associated with cellular membranes [4]. Sphingolipid metabolites play important roles in the regulation of glucose metabolism. SPK1 is the key enzyme in the sphingolipid metabolic pathway, forming an essential checkpoint to regulate the relative levels of bioactive sphingolipid metabolites, ceramide, sphingosine, and S1P. Previously [5], we demonstrated that adenovirusmediated SPK1 gene transfer (Ad-SPK1) improved blood glucose and lipid profiles, reduced body weight and adiposity, reversed hepatic steatosis, increased energy expenditure, and improved insulin sensitivity in diabetic mice. In this study, we investigated the effect of SPK1 on wound healing in diabetes mellitus.
http://dx.doi.org/10.1016/j.injury.2014.03.003 0020–1383/ß 2014 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Yu H, et al. Sphingosine kinase 1 improves cutaneous wound healing in diabetic rats. Injury (2014), http://dx.doi.org/10.1016/j.injury.2014.03.003
G Model
JINJ-5672; No. of Pages 5 2
H. Yu et al. / Injury, Int. J. Care Injured xxx (2014) xxx–xxx
SPK1 catalyses the phosphorylation of sphingosine to form S1P [6]. An increasing amount of evidence indicates that S1P/SPK plays a pivotal role in the signalling pathways of diverse cellular processes in various cell types, including proliferation, differentiation, migration, and apoptosis [7–10]. SPK1 also promotes cell growth in soft agar [11]. After demonstrating markedly improved blood glucose levels in type 2 diabetic KK/Ay mice by Ad-SPK1, we designed the present study to assess the effect of SPK1 on wound healing in diabetic rats.
after wounding. The wound size was then calculated by weighing the film. Animals were sacrificed 7, 14, and 21 days after wounding and skin specimens were obtained and fixed in 10% paraformaldehyde for histological study. Rate of wound healing
Materials and methods
The percentage of wound closure was calculated as follows, using the initial and final wound area drawn on glass slides during the experiments: % of wound healing = (original area unhealed area)/original area 100%.
Animals and plasmid
Histopathological assay
Healthy adult male Wistar rats (approximately 10 weeks of age; average body weight of 250–300 g) were used in this study. All rats were housed at a constant temperature and humidity in a room with an artificial 12-h light/dark cycle and had free access to food and water. Constructs of wild-type SPK1 (pcDNA3-FLAG-SPK1) were kindly provided by Dr. Stuart M. Pitson (Hanson Institute, Adelaide, Australia) [12]. All animal experiments were conducted following the institutional guidelines and approved by the Ethical Committee for Animal Care and Use, The Military Medical Science Academy of the PLA.
A skin specimen was obtained from each group on day 7, 14, 21 after wounding for histopathological examination. Specimens were immediately fixed in 10% (v/v) neutral buffered formalin, and the solution was replaced every 2 days until the tissues had hardened. Each specimen was embedded in a paraffin block and thin sections (3 mm) were prepared and stained with haematoxylin and eosin (H&E) for general morphological observation.
Reagent and antibody Streptozotocin (STZ) was purchased from Sigma (St. Louis, MO, USA). Rabbit anti-FLAG antibody was obtained from OriGene (Rockville, MD, USA). Induction of diabetes and wounding Forty male rats were used; five were randomly selected as normal controls. Diabetes was induced in the remaining rats by tail intravenous injection of the pancreatic b-cell toxin STZ (freshly dissolved in sterile saline, 2%) at a dose of 50 mg/kg body weight. Blood glucose levels were measured using an acute glucometer (Roche, USA) 1 week after the STZ injections. Animals that developed average blood glucose concentrations exceeding 18 mmol/L were selected and assigned randomly to four groups. Wounding was performed as described previously [13]. Briefly, rats were anaesthetised with intraperitoneal Nembutal (50 mg/ kg). The dorsal skin was shaved and cleaned with povidone-iodine solution. Two full-thickness skin wounds (approximately 1.8-cm diameter) were created on the back of each rat: the area was marked with a stamp before the outlined skin was cut. Therapeutic intervention and wound area Different concentrations of plasmid-SPK1 constructs (pcDNA3FLAG-SPK1; 15, 30, 60, 120 mg/wound) were applied to the wounds every 3 days. The construct was not applied to rats in the diabetic control group. Wound size was recorded with a transparent film after anaesthesia at 0, 3, 7, 10, 14, and 21 days
Immunohistochemistry Tissue sections that had been dewaxed and rehydrated routinely were incubated with 3% H2O2 for 30 min. The slides were washed with phosphate-buffered saline (PBS, pH 7.4) twice. The sections were blocked with 5% bovine serum albumin in Trisbuffered saline for 20 min. The spent solution was discarded and the sections were incubated with anti-FLAG antibody (1:2000) at 4 8C overnight. The slides were washed with PBS, incubated with rabbit secondary antibody (1:5000) for 1 h, and followed by incubation with streptavidin–horseradish peroxidase for 20 min. The antibody binding sites were visualised by incubation with diaminobenzidine–H2O2 solution. Statistical analysis All values are expressed as mean standard error of the mean (SEM). One-way analysis of variance followed by Student’s unpaired t-test was used to compare parametric data. P < 0.05 was recognised to indicate statistical significance. Results Effect of SPK1 on blood glucose levels of diabetic rats There was a significant elevation in blood glucose level (>18 mmol/L) after 1 week in the rats that had received STZ. Blood glucose levels were determined when the plasmid treatment was administered, as shown in Table 1; all diabetic rats had average blood glucose levels that exceeded 18 mmol/L, although there was no statistically significant difference among the six groups. Our data indicate that SPK1 did not directly affect the blood glucose levels of diabetic rats.
Table 1 Effect of SPK1 on blood glucose levels of diabetic rats. Group
Dose mg/wound
Times
Control Diabetic control SPK1 SPK1 SPK1 SPK1
Physiological saline Physiological saline 120 60 30 15
3 3 3 3 3 3
Blood glucose (mmol/L) 0d
7d
14 d
21 d
5.6 0.3 20.3 2.7 20.6 4.5 19.9 0.07 20.2 2.4 19.9 2.9
5.8 0.5 26.2 4.0 24.9 54.2 27.8 5.8 28.9 4.2 26.2 6.1
5.1 0.2 30.7 5.4 25.7 2.4 31.3 3.0 28.8 5.3 27.0 6.4
6.4 0.5 31.0 1.3 27.4 2.5 26.9 0.8 28.2 2.9 25.4 7.2
Please cite this article in press as: Yu H, et al. Sphingosine kinase 1 improves cutaneous wound healing in diabetic rats. Injury (2014), http://dx.doi.org/10.1016/j.injury.2014.03.003
G Model
JINJ-5672; No. of Pages 5 H. Yu et al. / Injury, Int. J. Care Injured xxx (2014) xxx–xxx
Fig. 1. Effect of SPK1 on wound areas of diabetic rats. Wound size was recorded with transparent film after anaesthesia at 0, 3, 7, 10, 14, and 21 days after wounding. The wound size was calculated by weighing the transparent films. Each bar represents the mean SEM wound area of each group. *P < 0.05, **P < 0.01, #P < 0.001 vs. diabetic control group.
Effect of SPK1 on wound areas of diabetic rats Wounds in all groups decreased in size gradually over time (Fig. 1). However, there was an obvious delay in the rate of healing in diabetic rats as compared with normal control rats. The wound areas of the diabetic rats were larger than that of the normal control rats after 3–21 days. Observation of the diabetic rats at 7 days revealed a statistically significant decrease in the wound area following treatment with 125 mg/wound (0.67 0.15 cm2, P < 0.001) and 60 mg/wound (0.83 0.18 cm2, P < 0.01) pcDNA3FLAG-SPK1 compared with diabetic control group (1.09 0.23 cm2). However, there was no significant change to the wound area at 3 days following treatment with 30 mg/wound pcDNA3-FLAG-SPK1 until day 7 of the treatment period, when a significant difference was observed (P < 0.05). No significant difference was detected when the dosage was reduced to 15 mg/wound. Effect of SPK1 on rate of wound healing in diabetic rats There was an obvious improvement in both wound area and rate of wound healing following treatment with different doses of pcDNA3-FLAG-SPK1 (Figs. 1 and 2). There was satisfactory healing following treatment with 125 and 60 mg/wound doses from 3 to 21 days. There was a statistically significant difference between the treatment and control groups (P < 0.05). After 21 days, the mean rate of healing was 98%. When a 30 mg/wound dose was administered, the initial effectiveness was delayed. When the dosage was reduced to 15 mg/wound, there was no effective healing. All groups had healed fully after 21 days. The results demonstrated that at a certain dosage range, SPK1 could accelerate wound healing in diabetic rats; the initial effective dose was 30 mg/ wound. A higher dose and earlier administration was followed by a higher rate of healing, which demonstrates an obvious dose–effect relationship. Effect of SPK1 on histopathological features of wounds of diabetic rats Following H&E staining (Fig. 3), the histopathological characteristics of the wounds on day 7, 14, and 21 after wounding were observed. After 7 days, control group specimens contained necrotic tissue and necrotic inflammatory cells, a small quantity of granulation tissue hyperplasia, and absence of re-epithelialisation (Fig. 3A). Conversely, tissue from the treatment groups (Fig. 3B) contained a large quantity of granulation tissue and abundant capillaries. After 14 days, the control group exhibited poor hyperplasia of granulation tissue accompanied by oedema and thinner re-epithelialisation at the edge of the wound with necrosis (Fig. 3C). Specimens from the treatment groups (Fig. 3D) contained
3
Fig. 2. Effect of SPK1 on rate of wound healing in diabetic rats. The percentage of wound closure was calculated by using the initial and final wound area as outlined on glass slides during the treatment period. Each bar represents the mean SEM wound healing rate of each group. *P < 0.05, **P < 0.01, #P < 0.001 vs. diabetic control group.
granulation tissue that had changed to fibrous connective tissue, and there were abundant capillaries. There was apparent reepithelialisation at the edge of the wound, which extended to cover the wound. After 21 days, control group specimens contained granulation tissue that had changed into fibrous connective tissue accompanied by oedema, and there was apparent re-epithelialisation that extended to cover the wound, but the tissue structure was incomplete (Fig. 3E). However, treatment groups specimens (Fig. 3F) showed that the granulation tissue had completely changed into fibrous connective tissue and the cutaneous wound had healed completely in most of the rats in the experiment. Detection of SPK1 expression in wound areas We detected the expression of SPK1 in the rat skins by immunohistochemistry (Fig. 4). Immunohistochemical staining with FLAG antibody demonstrated that there was high expression of SPK1 in the treatment groups, indicating that SPK1 was expressed on the surfaces of the wounds. Discussion Impaired wound healing, a common complication of diabetes mellitus, is characterised by diminished collagen production and impaired angiogenesis [14]. Wound healing is a complex multifactorial process involving inflammation, migration of different cell types, fibroplasia, collagen deposition, and wound contraction that results in the contraction and closure of the wound and restoration of a functional barrier [3]. Diabetes mellitus is one of the major contributors to chronic wound healing problems, as minor skin wounds can develop into chronic, nonhealing ulcers, ultimately resulting in infection, gangrene, or even amputation [3]. SPK is an enzyme that catalyses the phosphorylation of sphingosine to S1P. Zhang et al. demonstrated that exogenously applied sphingosine is rapidly taken up by cells and phosphorylated on its primary hydroxyl group into S1P [15]. Exogenous sphingosine can stimulate cell proliferation in several cell types [16,17]. This mitogenic effect of sphingosine may be mediated by its conversion to S1P, as S1P itself is an even more potent mitogen than sphingosine [15]. S1P is a sphingolipid metabolite present in high concentrations in the blood and is implicated in vascular development. It is secreted most prominently by platelets, suggesting that it plays an important role in tissue repair. Further, it has been shown to act via distinct receptor pathways to regulate keratinocyte and fibroblast chemotaxis, processes that are critical for normal wound healing [18,19]. As proof of concept,
Please cite this article in press as: Yu H, et al. Sphingosine kinase 1 improves cutaneous wound healing in diabetic rats. Injury (2014), http://dx.doi.org/10.1016/j.injury.2014.03.003
G Model
JINJ-5672; No. of Pages 5 4
H. Yu et al. / Injury, Int. J. Care Injured xxx (2014) xxx–xxx
Fig. 3. Histological examination of H&E-stained tissue sections. Photomicrographs depict tissue sections of the wounds on rats in the control and treatment groups on days 7, 14, and 21 after wounding. (A) Control group after 7 days; (B) treatment group after 7 days; (C) control group after 14 days; (D) treatment group after 14 days; (E) control group after 21 days; (F) treatment group after 21 days (HE stain, 200).
Fig. 4. Expression of SPK1 in cutaneous wounds of diabetic rats on day 7: (A) control group and (B) treatment group (200 magnification).
subcutaneous injections of S1P significantly improved diabetic wound healing and neovascularisation in rodent models [20]. We constructed a plasmid containing SPK1 cDNA and applied it to wound surfaces to examine its effect on wound healing, and found that SPK1 promoted wound healing. We hypothesised that SPK1 catalysed the phosphorylation of sphingosine to S1P in the skin. It is possible that this is a new approach to cutaneous wound repair. It is noteworthy that enhanced wound healing by
SPK1 cDNA application in diabetic rats is independent of blood glucose control. As shown in Table 1, there was no statistically significant difference in the blood glucose levels between the treated and control diabetic animals. Cutaneous wound healing is a complex pathological process in which a number of tissues, extracellular matrix, and cytokines are involved, and includes a series of biological processes. A key event in the process is hyperplasia of granulation tissue. The formation of
Please cite this article in press as: Yu H, et al. Sphingosine kinase 1 improves cutaneous wound healing in diabetic rats. Injury (2014), http://dx.doi.org/10.1016/j.injury.2014.03.003
G Model
JINJ-5672; No. of Pages 5 H. Yu et al. / Injury, Int. J. Care Injured xxx (2014) xxx–xxx
granulation tissue in a wound can repair tissue defects, enhance resistance of the wound to infection, and promote contraction so that the wound heals, creating the necessary conditions for epithelial climb. Granulation tissue is mainly composed of fibroblasts and new capillaries. Angiogenesis plays an essential role in the processes of organisation healing [21]. The proliferation of capillary endothelial cells and fibroblast cells can significantly promote the proliferation of granulation tissue and secrete many functional proteins while promoting wound contraction and expansion. The quantity of capillaries and fibroblasts determine the duration of wound healing. In the present study, histopathological examination revealed rapid re-epithelialisation and promotion of granulation tissue as well as blood vessel growth following the application of plasmids that carried the SPK1 gene to the skin around the wound. Located in the dermis layer, fibroblasts are one of the major repair cells. Wound repair depends on the biological behaviour of fibroblasts. The process of wound healing is a highly orchestrated event that entails the release of proinflammatory factors and growth factors. Once injury occurs, fibroblasts activate and proliferate in the early stages after wounding, becoming myofibroblasts which express smooth muscle actin (SMA). These newly formed cells participate in the secretion of many factors, wound contraction, and extracellular matrix production. With continuous wound repair, SMA expression is decreased and myofibroblasts gradually disappear, and the expression of various growth factors gradually decreases [22,23]. Therefore, myofibroblasts play a central role in wound healing. The involvement of SPK1 in wound healing effected by fibroblasts requires further investigation. Conclusions SPK1 had an obvious effect on wound healing in diabetes mellitus, accelerating wound healing in diabetic rats. There was an obvious improvement in both wound area and rate of wound healing following treatment with different doses of pcDNA3-FLAGSPK1. However, the treatment did not affect the blood glucose levels of the diabetic rats. Conflict of interest statement There are no conflicts of interest. Acknowledgements We thank Yonghong Lei for his help with data analysis. We thank Xiaoyu Zhu for her valuable help in the last phase of this study.
5
References [1] Leahy JL. Pathogenesis of type 2 diabetes mellitus. Arch Med Res 2005;36:197– 209. [2] Jahangiri Noudeh Y, Shabani M, Vatankhah N, Hashemian SJ, Akbari K. A combination of 670 nm and 810 nm diode lasers for wound healing acceleration in diabetic rats. Photomed Laser Surg 2010;28:621–7. [3] Lee YH, Chang JJ, Chien CT, Yang MC, Chien HF. Antioxidant sol–gel improves cutaneous wound healing in streptozotocin-induced diabetic rats. Exp Diabetes Res 2012;2012:504693. [4] Spiegel S, Milstien S. Sphingosine-1-phosphate: an enigmatic signalling lipid. Nat Rev Mol Cell Biol 2003;4:397–407. [5] Duan HF, Wu CT, Lu Y, Wang H, Liu HJ, Zhang QW, et al. Sphingosine kinase activation regulates hepatocyte growth factor induced migration of endothelial cells. Exp Cell Res 2004;298:593–601. [6] Nava VE, Lacana E, Poulton S, Liu H, Sugiura M, Kono K, et al. Functional characterization of human sphingosine kinase-1. FEBS Lett 2000;473:81–4. [7] Pyne S, Pyne NJ. Sphingosine 1-phosphate signalling in mammalian cells. Biochem J 2000;349:385–402. [8] Xia P, Gamble JR, Wang L, Pitson SM, Moretti PA, Wattenberg BW, et al. An oncogenic role of sphingosine kinase. Curr Biol 2000;10:1527–30. [9] Spiegel S, Milstien S. Sphingosine 1-phosphate, a key cell signaling molecule. J Biol Chem 2002;277:25851–54. [10] Olivera A, Kohama T, Edsall L, Nava V, Cuvillier O, Poulton S, et al. Sphingosine kinase expression increases intracellular sphingosine-1-phosphate and promotes cell growth and survival. J Cell Biol 1999;147:545–58. [11] Olivera A, Spiegel S. Sphingosine kinase: a mediator of vital cellular functions. Prostaglandins 2001;64:123–34. [12] Pitson SM, Moretti PA, Zebol JR, Xia P, Gamble JR, Vadas MA, et al. Expression of a catalytically inactive sphingosine kinase mutant blocks agonist-induced sphingosine kinase activation. A dominant-negative sphingosine kinase. J Biol Chem 2000;275:33945–50. [13] Mori R, Kondo T, Ohshima T, Ishida Y, Mukaida N. Accelerated wound healing in tumor necrosis factor receptor p55-deficient mice with reduced leukocyte infiltration. FASEB J 2002;16:963–74. [14] Hansen SL, Myers CA, Charboneau A, Young DM, Boudreau N. HoxD3 accelerates wound healing in diabetic mice. Am J Pathol 2003;163:2421–31. [15] Zhang H, Desai NN, Olivera A, Seki T, Brooker G, Spiegel S. Sphingosine-1phosphate, a novel lipid, involved in cellular proliferation. J Cell Biol 1991;114:155–67. [16] Zhang H, Buckley NE, Gibson K, Spiegel S. Sphingosine stimulates cellular proliferation via a protein kinase C-independent pathway. J Biol Chem 1990;265:76–81. [17] Merrill Jr AH, Schmelz EM, Dillehay DL, Spiegel S, Shayman JA, Schroeder JJ, et al. Sphingolipids – the enigmatic lipid class: biochemistry, physiology, and pathophysiology. Toxicol Appl Pharmacol 1997;142:208–25. [18] Estrada R, Zeng Q, Lu H, Sarojini H, Lee JF, Mathis SP, et al. Up-regulating sphingosine 1-phosphate receptor-2 signaling impairs chemotactic, woundhealing, and morphogenetic responses in senescent endothelial cells. J Biol Chem 2008;283:30363–75. [19] Vogler R, Sauer B, Kim DS, Schafer-Korting M, Kleuser B. Sphingosine-1phosphate and its potentially paradoxical effects on critical parameters of cutaneous wound healing. J Invest Dermatol 2003;120:693–700. [20] Kawanabe T, Kawakami T, Yatomi Y, Shimada S, Soma Y. Sphingosine 1phosphate accelerates wound healing in diabetic mice. J Dermatol Sci 2007;48:53–60. [21] Isner JM, Losordo DW. Therapeutic angiogenesis for heart failure. Nat Med 1999;5:491–2. [22] Powell DW, Mifflin RC, Valentich JD, Crowe SE, Saada JI, West AB, et al. Paracrine cells important in health and disease. Am J Physiol 1999;277:C1–9. [23] Badid C, Vincent M, Fouque D, Laville M, Desmouliere A. Myofibroblast: a prognostic marker and target cell in progressive renal disease. Ren Fail 2001;23:543–9.
Please cite this article in press as: Yu H, et al. Sphingosine kinase 1 improves cutaneous wound healing in diabetic rats. Injury (2014), http://dx.doi.org/10.1016/j.injury.2014.03.003
